S. mansoni before treatment (G-II) and infected by S. mansoni after treatment (G-III). Renal function was evaluated by tubular and glomerular biomarkers and through urinary MCP-1. Patients’ mean age was 23.2±13 years. There was no statistically significant difference between the groups regarding tubular and glomerular function evaluated through the traditional biomarkers. MCP-1 was higher in G-II and G-III, when compared to G-I; p=0.009 and p=0.007, respectively. There was no difference when comparing groups G-II and G-III (p=0.892). Although it was not different among the groups, there was a significant correlation between albuminuria and MCP-1. There was a significant increase in urinary MCP-1 levels in patients with schistosomiasis mansoni, which was associated with albuminuria. This protein has a role in the recruitment of monocytes to injury and inflammation sites . The increase of MCP-1 in the urine evidences that there is silent renal inflammation in these patients and the inflammatory status is not interrupted by specific treatment of the offending agent. Our findings suggest that urinary MCP-1 can be a sensitive marker of renal injury in patients with schistosomiasis mansoni. The aim of this study is to investigate renal markers and the biomarker MCP-1 in patients with schistosomiasis mansoni. This is a cross-sectional study with 85 patients aged 5 to 48 years, with a confirmed diagnosis of schistosomiasis mansoni through the Kato-Katz method. The patients were divided in three groups: control (G-I); infected by  Schistosoma mansoni can lead to slow and progressive renal damage in addition to membranoproliferative glomerulonephritis (MPGN).The association between infectious endemic diseases and the kidney has already been described . Renal iRoutine markers for renal damage  lack sensitivity. Even the use of microalbuminuria, one of the most sensitive markers of renal damage currently used in clinical practice is controversial and some studies have suggested that higher normal range values can be related to progressive renal damage . RecentlTo date, there has been no study regarding renal involvement in patients with chronic schistosomiasis mansoni. The aim of this study is to investigate renal markers and the biomarker MCP-1 in patients with schistosomiasis mansoni.In compliance with the ethical rules governing research on human health provided in Resolution 196/96 of the National Health Council of Brazil, this project was submitted to the Research Ethical Committee of the Department of Physiology and Pharmacology of Universidade Federal do Ceara and approved under n. 165/09. Before selection, all families attended a lecture detailing the objectives and procedures of this study. Those who chose to participate had the study explained in full at home. Informed written consent was obtained from the study participants or, in the case of minors, from their parents or guardians.Biomphalaria straminea is the intermediate host of schistosomiasis mansoni. Patients older than 60 and younger than 2 were excluded, as well as those with diabetes mellitus, hypertension, systemic lupus erythematosus, rheumatoid arthritis, chronic kidney disease and any other condition that could cause kidney injury. Moreover, a urine microscopy study was performed to exclude significant leukocyturia (higher than 5 white blood cells per high-power field). A total of 85 individuals were selected, according their status for S. mansoni infection. Group I (GI): non-infected subjects, demonstrated by negative serology through ELISA and Kato-Katz methods - n=24; Group II (GII); infected subjects with no previous treatment (n=30) and group III (G III) infected subjects treated between 6-12 months before study inclusion. In the last group, reinfection was excluded by performing new Kato-Katz test (3 samples) at study inclusion. This is a cross-sectional study carried out in Planalto do Cajueiro, a district in the town of Maranguape, Ceara State, Northeast of Brazil. This district is a small community distributed across five urban blocks, according to a survey carried out by the town hall. The area is surrounded by two streams and S. mansoni. After this analysis, feces were collected for Kato-Katz testing [Blood samples were collected to perform sorting of individuals by ELISA using soluble adult worm antigen (SWAP) of  testing , to dete® kits and the results were used to estimate fractional excretion. Urinary MCP-1 measurement was performed through ELISA using the R&D Systems® kit, Inc . The MCP-1 value was corrected by urinary creatinine.Serum creatinine and urinary albumin were determined by the kinetic-colorimetric method and the results were expressed in mg/dL. After the sample analysis, the eGFR was calculated by the Cockcroft-Gault  and SchwS. mansoni eggs in the stool were treated with Praziquantel® at a dose of 60 mg/kg of body weight in a single dose. The drug was dispensed under medical supervision at the community’s public health center. Patients in the GIII had their treatment efficacy and no reinfection confirmed by a new Kato-Katz test.All patients who were positive for the presence of Data were expressed as mean ± standard deviation. Statistical analysis was carried out using the SPSS program, version 19.0 . After testing for normality using the K-S test, differences between the means of multiple variables were inferred trough One-Way ANOVA test and Bonferroni post-test and the correlations were evaluated according to Pearson’s test. The established confidence interval was 95%, and “p” values <0.05 were considered statistically significant.Patients’ mean age was 23.2 ± 13 years. There was no significant difference regarding age (p=0.50) and gender (p=0.18) among the groups.The cut-off value used for ELISA was 0.283 (±2SD). The mean reading of ELISA in groups I, II, III  were, respectively, 0.18, 0.48 and 0.62. None of ELISA tests were performed after treatment.S. mansoni eggs in all cases and 3 cases also had other parasitic diseases, while in the post-treatment group (GIII), only 1 patient had other parasitic infection, with Trichuris trichiura being the other parasite found.In the infected group (GII), there were There was no difference regarding tubular function among the three groups . RegardiMCP-1 levels were significantly higher in GII (178 ± 97pg/mg-Cr) and GIII (175 ± 87pg/mg-Cr), when compared to GI (123 ± 48pg/mg-Cr), p=0.009 and p=0.007, respectively, but there was no difference when comparing GII and GIII (p=0.892) . Althoug Although the intensity of the humoral response to the parasite can cause some damage by the immune complexes deposition, we found no correlation among IgG-ELISA and the markers used as MCP-1 , albuminuria  and GFR .For the first time, we have shown that schistosomiasis mansoni infection can induce a chronic renal inflammatory status, demonstrated by an increment in urinary MCP-1. However, this inflammatory status is not interrupted by the specific treatment of the offending agent.Most patients in the present study were young with low probability of underlying kidney disease. Although there was no difference among the three groups in relation to urinary albumin excretion rate, an increase in urinary MCP-1 was observed in patients with active or treated schistosomiasis mansoni infection. This finding suggests that urinary MCP-1 may be a more sensitive marker of renal damage than urinary albumin excretion rate.In other renal diseases, urinary MCP-1 has been correlated with urinary albumin excretion rate, glomerular filtration rate reduction and other features of renal injury. To date, studies have included metabolic (diabetes mellitus), immunologic (lupus nephritis) and genetic  kidney disorders ,13. To tAnother marker of glomerular kidney injury that has been studied is cystatin C. It is an endogenous marker of renal dysfunction; in fact, it is a more adequate marker of GFR, rather than a primary AKI biomarker. Furthermore, there are also tubular biomarkers such as Kidney Injury Molecule-1(KIM-1), Neutrophil gelatinase-associated lipocalin (NGAL), Interleukin-18 and N-acetyl-β-D-glucosaminidase (NAG). KIM-1and NGAL have been studied in humans and rodents. KIM-1 is not detectable in normal kidney tissue or urine, but is expressed at very high levels in kidney tubular epithelial cells of rodents and humans after ischemic injury and NGAL is expressed by neutrophils and epithelial cells including those of the proximal tubule .Trypanosoma cruzi Y strain [In a recent study, kidney injury was characterized by high levels of proteinuria, Kidney Injury Molecule-1 (KIM-1) and urea, in guinea pigs infected and non-infected with Y strain .It is noteworthy the fact that even 6 months after schistosomiasis mansoni treatment, there was no reduction in urinary MCP-1. Although most patients had albumin excretion rate within the normal range, a positive and significant correlation was observed between albuminuria and urinary MCP-1. It is already know that even patients with high-normal albumin excretion rate are at high-risk of developing chronic renal failure. So, this study suggest that young patient with schistosomiasis mansoni infection are at higher risk to develop clinically significant kidney injury than elder patients.S. mansoni and this correlated with albuminuria levels, which were within normal limits. As schistosomiasis mansoni is a disease that predominates in youngerindividuals, these patients are at higher risk of renal function loss and should benefit from measures to slow kidney disease progression. MCP-1 seems to be a useful early biomarker for schistosomal nephropathy.These data strongly suggests that there was a chronic and asymptomatic inflammation in these patients infected with


Haemonchus contortus; in many parts of the world this species is resistant to almost all the currently available drugs, including ivermectin. It is extremely polymorphic and to date it has proved impossible to relate any sequence polymorphisms to its ivermectin resistance status. Expression of candidate drug-resistance genes in Caenorhabditis elegans could provide a convenient means to study the effects of polymorphisms found in resistant parasites, but may be complicated by differences between the gene families of target and model organisms. We tested this using the glutamate-gated chloride channel (GluCl) gene family, which forms the ivermectin drug target and are candidate resistance genes. We expressed GluCl subunits from C. elegans and H. contortus in a highly resistant triple mutant C. elegans strain (DA1316) under the control of the avr-14 promoter; expression of GFP behind this promoter recapitulated the pattern previously reported for avr-14. Expression of ivermectin-sensitive subunits from both species restored drug sensitivity to transgenic worms, though some quantitative differences were noted between lines. Expression of an ivermectin-insensitive subunit, Hco-GLC-2, had no effect on drug sensitivity. Expression of a previously uncharacterised parasite-specific subunit, Hco-GLC-6, caused the transgenic worms to become ivermectin sensitive, suggesting that this subunit also encodes a GluCl that responds to the drug. These results demonstrate that both orthologous and paralogous subunits from C. elegans and H. contortus are able to rescue the ivermectin sensitivity of mutant C. elegans, though some quantitative differences were observed between transgenic lines in some assays. C. elegans is a suitable system for studying parasitic nematode genes that may be involved in drug resistance.Anthelmintic resistance is a major problem in livestock farming, especially of small ruminants, but our understanding of it has been limited by the difficulty in carrying out functional genetic studies on parasitic nematodes. An important nematode infecting sheep and goats is  Caenorhabditis elegans, for such studies, taking advantage of the powerful genetic and behavioural tools that can be applied to this organismHaemonchus contortus is an economically important parasite of small ruminants which has developed widespread resistance to anthelmintic drugs, including the MLs Infections with parasitic nematodes are a major problem in animal health, and for human health in less-developed parts of the world Strongyloides stercoralis has been successfully transformed H. contortus has not been successfully used in studies of transgenesis so far, although RNAi may be useful in the future for studying genes in H. contortus by gene knockdown C. elegans, our aim was to exploit C. elegans for the study of H. contortus GluCl subunit function in vivo. Successful expression of subunits from the parasite in C. elegans would enable us to interrogate the function of particular GluCl subunits.Investigation of the behavioural and drug target roles of individual GluCl subunits in parasitic nematodes is hampered by several limitations. The requirement for a host to complete the lifecycle of the parasite is a major obstacle in producing mutant strains of parasitic nematode required for a detailed analysis of GluCl subunit function. Transgenesis of parasitic nematodes is possible C. elegans. Gillan et al., studying hsp-90, found that the H. contortus orthologue produced only a partial rescue of the C. elegans mutation, whereas the orthologue from Brugia pahangi, a more distantly related parasite, failed to produce any rescue H. contortus genes that have been successfully expressed in C. elegans include cathepsin L elt-2C. elegans to become drug resistant. Genes from other parasites have also been expressed; Kampkotter et al Onchocerca volvulus would make C. elegans more resistant to oxidative stress and Massey et al. Strongyloides stercoralis would rescue daf-16 associated defects. In contrast, Crook et al. daf-7 orthologue from Parastrongyloides trichosuri  under the control of the avr-14 promoter. Transformation with any of these cDNAs resulted in rescue of ivermectin sensitivity to varying degrees  that is only weakly resistant to ivermectin Cel-avr-14a cDNA in DA1316 showed different levels of ivermectin sensitivity, from 33±8.6% of line 2 paralysed to 78±11.8% of line 1; the differences between avr-14A lines 2 & 3, and the avr-14B lines were significant (p = <0.01).The movement of the triple GluCl mutant strain, DA1316, was not affected by exposure to 1 µM ivermectin after 1 hour, in contrast to the total paralysis of 100% of wild type nditions . In orde degrees , with stC. elegans with mutations in the three GluCl subunit genes reduced their thrashing rate by only 50%. In contrast, the transformation of DA1316 with avr-14b cDNA from either C. elegans or H. contortus resulted in an increase in the sensitivity of these worms to ivermectin to such an extent that they had a thrashing rate of only 2±1 and 2±2 thrashes/min respectively at 10 µM drug, and their thrashing rate was reduced by more than 50% at 1 µM ivermectin. The results from transgenic worms expressing Cel-AVR-14A were more variable, with two out of three lines retaining some activity in 1 µM ivermectin  and pharyngeal pumping activity in different lines of C. elegans. Control plates containing 0.1% (v/v) DMSO were also set up during each experiment to account for any non-ivermectin related effects. The triple mutant line was resistant to ivermectin throughout the concentration range used, whereas no wild type adults were observed at concentrations above 1 nM ivermectin  also reversed at the same frequency as the wild-type worms, adding additional support for this suggestion. The expression of Cel-AVR-14B had no effect on the reversal frequency of DA1316, but transforming the triple mutant with Hco-avr-14b cDNA produced worms with a severely reduced ability to reverse at normal frequencies.Our previous study had indicated that mutations in GluCl subunits affected the reversal frequency of 14b cDNA . The revC. elegans and H. contortus and the subunit composition of the native channels is not known. In vitro, Cel-GLC-1 and -GLC-2 can co-assemble to form functional channels in vivo, Cel-glc-2 is expressed only in the pharynx Cel-avr-15 is also expressed Cel-glc-1 has not yet been determined. In H. contortus, the expression patterns of Hco-AVR-14 and Hco-GLC-5 overlap in vivo, AVR-14 subunits co-assemble with others to form ivermectin-sensitive heteromeric channels, this might mean that expression of non-orthologous GluCl genes would not rescue the ivermectin-resistance phenotype effectively, perhaps limiting the usefulness of this approach for studying genes from other species. We therefore transfected DA1316 with cDNAs encoding other ivermectin-sensitive subunits from C. elegans  and H. contortus (Hco-glc-5). We also used cDNA encoding an ivermectin-insensitive subunit, Hco-GLC-2 H. contortus. All of the cDNAs were sub-cloned behind the same avr-14 promoter used previously. The results, shown in C. elegans subunits, all three lines expressing Cel-AVR-15 were almost completely sensitive to 1 µM ivermectin, whereas those expressing Cel-GLC-3 or Cel-GLC-1 were only partially sensitive to this concentration of the drug. Expression of the H. contortus GLC-5 subunit also caused a partial rescue of the sensitivity to 1 µM ivermectin, and complete sensitivity to 10 µM, whereas expression of Hco-GLC-2 did not produce any significant changes to DA1316, which is consistent with the insensitivity of GLC-2 channels to ivermectin The GluCl are pentameric channels, there are multiple GluCl subunit genes in both in vitro studies are useful, especially in the case of those drugs which affect ion channels, in vivo confirmation that a particular sequence change does alter the organism's response to drug application is always desirable. C. elegans has been used as an expression system for many parasite genes, including those involved in resistance to the benzimidazole anthelmintics avr-14, being widely conserved and others seeming to be present in much fewer species C. elegans, as is the case for H. contortus, raises some interesting problems, especially if they are required to assemble with other subunits to form heteromeric receptors in order to be fully functional.The study of polymorphisms and mutations potentially involved in anthelmintic resistance of parasitic nematodes is complicated by the difficulty of carrying genetic experiments on these organisms. Though C. elegans and H. contortus in a C. elegans strain (DA1316) that is very resistant to ivermectin, and by testing the sensitivity of the transgenic worms to the drug. DA1316 is a triple mutant strain  but the double mutant  has a very low-level of resistance avr-14 mutation would produce a clear shift in response to the drug. A rescue of ivermectin sensitivity implies that the recombinant subunit forms, either by itself or by assembling with other endogenous polypeptides, a functional drug-sensitive channel. We chose H. contortus as the source of the parasite cDNAs for this study because it is a member of clade V like C. elegans, and is therefore not too phylogenetically distant C. elegans genome  than on Cel-AVR-14B (EC50 = 2.2 mM) that we observed in vitroglc-1, which must be able to suppress the increase in reversal frequency caused by the avr-14 and avr-15 mutations. We have previously shown that not all mutation in GluCl genes cause an increase in reversal frequency; a deletion in glc-3 causes a marked decrease in reversal frequency avr-15 and glc-1. This strain showed no abnormality in reversal frequency  agar plates seeded with OP50 bacteria.C. elegans and H. contortus GluCl cDNA fragments were cloned behind a Cel-avr-14 promoter fragment, as described previously avr-14 promoter was a 1700 bp fragment from immediately upstream of the avr-14 translational start site. To test the expression pattern of this promoter fragment, a transcriptional GFP reporter construct was made by cloning the avr-14 promoter fragment into a vector containing GFP  C. elegans and the expression pattern examined   . The seqC. elegans was carried out using DNA-coated gold particle bombardment (Bio-Rad PDS-1000 with hepta adaptor for increased transformation efficiency). For the rescue experiments, the DA1316 strain  and the test DNA were on separate plasmids, which were linearized by digestion with ApaI or KpnI in standard buffers (NEB) in a total reaction volume of 50 µl for each plasmid. The DNA solutions were then mixed together and precipitated onto gold particles. Transformation was carried out under 1500 psi of helium gas pressure and a chamber vacuum of 21 mmHg. Subsequently, transformants expressing GFP in the body wall muscle were picked and successful lines were maintained. The presence of the test DNA was verified for each line using single worm PCR, with primers either specific to the start and end of the cDNA sequence (for H. contortus cDNA) or by designing primers that spanned exon-exon junctions (for C. elegans cDNA). In practise, 100% of lines expressing the myo-3::gfp construct contained the test DNA. At least two separate lines were produced for each GluCl cDNA tested. A control line was produced by transforming DA1316 with the myo-3::gfp construct only, and this line is used in place of untransformed DA1316 throughout the rest of the assays.Transformation of A survey of the ivermectin sensitivity of the multiple lines was carried out for all worm strains produced. Ten individual worms were picked from each line/strain into 96-well plates with wells containing 50 µl M9 salts, two wells per line. 50 µl of 2 µM ivermectin solution was added to each well to produce a final concentration of 1 µM. The worms were left at 19°C for 1 h, after which time the number of stationary worms in each well was counted. The experiments were carried out in triplicate and using the data from this assay, one representative line containing each construct was chosen for use in the thrashing assay, chronic ivermectin sensitivity assay and behavioural assay.avr-14B transgenic lines more fully. The thrashing assay was carried out using two different methods: manual thrashing assay and automated thrashing assay. The two methods were used to verify the measurements thoroughly.Thrashing assays were carried out at different concentrations of ivermectin to characterise the drug sensitivity of the C. elegans were placed in 100 µl of ivermectin solution (made up in M9 buffer) in a 96-well microtitre plate. After 30 minutes in the drug at 19°C, the number of thrashes (bends of the body from one side to the other and back) were counted for 1 minute. A preliminary time-course investigation (data not shown) indicated that 30 minutes would be the appropriate duration in the drug at the ivermectin concentrations used (0.03 to 8.75 µg/ml). The experiment was carried out for 8 worms per strain, per ivermectin concentration and was repeated on two further days. The GluCl-transformed lines were chosen as representative lines, after inspection of the motility assay data. One thrash was counted as a movement of the head to one side e.g. a thrash was counted every time the head of the worm moved to the left.For the manual thrashing assay, individual young adult The automated thrashing assay was carried out as for manual thrashing, except that the thrashing rates for all worms could be measured simultaneously. Movies were taken of the whole 96-well plate, and thrashing rates calculated for each well by the co-variance method as described previously C. elegans lines were made, and eggs were transferred onto the ivermectin/DMSO plates. The number of adult worms in each well was counted six days later. Three replicate wells per ivermectin/DMSO concentration per worm strain were set-up, and the whole experiment was repeated on two subsequent occasions.NGM was made, allowed to cool, and spiked to contain a final concentration of either 0.01% DMSO or ivermectin at 100 pM or 1, 3.2, 10, or 100 nM. The NGM was transferred to 12-well plates, allowed to set and seeded with 50 µl OP50. After 2 days at room temperature, egg preparations of the different The frequency of reversals was as described by Cook et al


Melasma is a common hypermelanosis of the face. The use of a classical Q-switched ruby laser (QSRL) to treat melasma is discussed controversially and is associated with frequent adverse effects, such as hyper- or hypopigmentation. Recently a fractional-mode (FRx) QSRL was developed to minimize the adverse effects of classical QSRL. The objective of this research was to evaluate the efficacy and safety of a novel FRx-QSRL in the treatment of melasma in Caucasian patients.2. Three blinded investigators independently evaluated the melasma area and severity index (MASI) score before treatment and at the four- to six-week follow-ups. At additional three-month follow-ups, patients evaluated subjective improvement, pain and over-all satisfaction with the treatment according to a numeric analogue score (NAS). Side effects were documented.We performed a retrospective study of 25 Caucasian melasma patients (Fitzpatrick skin types I to III). Patients received one to three FRx-QSRL treatments  at pulse energies of 4 to 8 J/cmP = 0.0001) reduction of the MASI score from 6.54 to 1.98 (72.3%). Patients rated the pain of the intervention at a mean 2.46 points , the improvement at a mean 5.55 points  and the overall satisfaction at a mean 4.66 points . After three months, post-inflammatory hyperpigmentation (PIH) and/or recurring melasma were observed in 7 (28%) and 11 (44%) patients, respectively.At four to six weeks post laser treatment for a mean of 1.4 sessions, we observed a significant (The 694-nm FRx-QSRL is a safe and effective option for treating melasma in Caucasian patients. Over periods of >3 months, PIH and/or recurring melasma may develop at significant rates and may reduce patient satisfaction. Multiple treatment sessions with lower pulse energies and/or a post-interventional therapy with hypopigmenting ointments and UV protection may help to minimize these complications. Melasma is a common acquired benign pigmentary disorder that classically manifests as symmetrical hypermelanosis of the face. Most commonly melasma affects women of reproductive age with darker skin types (Fitzpatrick skin types IV and higher) but it may also occur at other ages and in men. Briefly, the Fitzpatrick skin type classification system is based on the color of the skin (type I being the fairest (white) and type VI being the darkest (black)) and its response to UV exposure (pigmentation) . An estiThe pathogenesis of melasma has remained largely elusive. Established concepts propose a stimulation of pigment-producing cells (melanocytes) by sex hormones (estrogen and progesterone) and UV irradiation. Recent studies have reported paracrine effects amongst melanocytes, keratinocytes and/or fibroblasts -7, and h2) lasers and intense pulsed light (IPL), have been evaluated for their efficacy in treating melasma [2) for treating melasma has been reported to be associated with a significant frequency of post-interventional hyper- as well as hypopigmentation [et al. demonstrated that melasma patients developed post-inflammatory hyperpigmentation (PIH) and worsening of the melasma after QSRL therapy [et al. reported that a novel fractional-mode (FRx) QSRL  may be effective in treating melasma in Korean patients [Various laser and light systems, including ruby lasers, Er:YAG lasers, carbon dioxide  and was initially presented by Manstein and coworkers in 2004 . The sigt lasers . Recentln = 14), the cheeks (n = 12), the chin (n = 2) or the upper lip (n = 2). Identified risk factors included past pregnancies (n = 5), hormonal therapy (n = 12) and UV exposure (n = 25). Some patients presented with melasma in multiple locations and/or risk factors.In the study, 25 Caucasian women  with melasma were treated with an FRx-QSRL  at the Medical Skin Center, Düsseldorf, in 2010 and 2011. Patients presented with epidermal or mixed-type melasma. Of the patients, 19 had had melasma <10 years and 6 had had it >10 years. The melasma areas were located on the forehead . Each patient received laser treatment for all affected areas. In pain-sensitive patients, a topical anesthetic (a lidocaine-tetracaine mix) was applied onto the treated areas 30 minutes prior to treatment. However, most patients tolerated the intervention without anesthesia. The laser treatment lasted up to 10 minutes depending on the size and number of treated areas. Post-intervention, cool packs were applied. Patients were advised to use strict UV protection. If more than one treatment was needed, the patient received additional treatments after intervals of four weeks.We used a 694-nm FRx-QSRL  at fluences of 4 to 8 J/cmPhotographs  were taken before therapy and at the four- to six-week follow-up sessions. Three blinded investigators independently evaluated the melasma area and severity index (MASI) score before treatment and at the four- to six-week follow-ups, as previously described . At the P < 0.05 was regarded as statistically significant.Student’s T-test was used for the statistical analysis, and The analysis included 25 patients. At the follow-up at four to six weeks after the last treatment (there were up to three treatment sessions with a mean of 1.4 sessions) the average MASI score showed a significant reduction from 6.54 to 1.98 (72.3%) , the improvement at a mean 5.55 points  and the overall satisfaction at a mean 4.66 points . We also used the same protocol to treat patients with darker skin types and melasma and/or PIH, with satisfactory results Figure .Laser therapy is based on the biophysical principle of selective photothermolysis. The lasers used emit light at a wavelength that is specifically and adequately absorbed by the target chromophores . The taret al. of 50 patients (Fitzpatrick skin types IV to VI) demonstrated a mean decrease in the MASI score of 61.3% after nine sessions. At the three-month follow-up a recurrence rate of 64% was noted [et al. reported improvement rates of 50% to 74% [et al. demonstrated a mean decrease of the MASI score of 29.8% in 15 Korean patients after six treatments with an FRx-QSRL . Two patients reported a slight worsening. None of the patients reported any long-term adverse effects such as PIH [et al. found that a QSRL was more effective for treating melasma compared to a QS-Nd:YAG laser [QS-Nd:YAG lasers have been shown to be effective and are widely used in the management of melasma -29. A re% to 74% . For the% to 74% ,20. Convh as PIH . Jang etAG laser . This haAG laser ,30.et al., we have demonstrated a significant (P = 0.0001) decrease of the MASI score of 72.3% in 25 Caucasian patients after a mean of 1.4 sessions with an FRx-QSRL . As the risk of common adverse effects of QSRL therapy, such as PIH, is expected to be significantly lower in Caucasian compared to Asian skin types, we applied higher fluences (4 to 8 J/cm2) per session compared to Jang et al. (2 to 3 J/cm2). However, this more aggressive treatment resulted in a significantly higher rate of adverse effects (PIH and/or recurring melasma in 28% and 44% of the patients). Lower fluences and multiple treatment sessions may reduce the frequency of these adverse effects. However, many patients refuse further treatment  if the result of an initial treatment is minimal. An additional option could be a multimodal therapy using an FRx-QSRL in combination with microdermabrasion [Consistent with the results of Jang abrasion , chemicaabrasion  and/or habrasion .In summary, the FRx-QSRL is a safe and effective treatment for melasma for patients with Caucasian or Asian skin types. Multimodal concepts may further increase the efficacy and reduce the adverse effects of the therapy. We maintained only a three-month follow-up after the final treatment, so further long-term studies are needed.Written informed consent was obtained from the patients for the publication of this report and any accompanying images.FRx: Fractional-mode; IPL: Intense pulsed light; LSR: Laser resurfacing; MASI: Melasma area and severity index; PIH: Post-inflammatory hyperpigmentation; QS: Q-switched; QSRL: Q-switched ruby laser; SCF: Stem cell factor.PA Gerber received honoraria for oral presentations by Asclepion Laser Technologies.SH and HH performed the laser treatments and collected the data. BAB, HS, EB and PAG evaluated and analyzed the data, and wrote the manuscript. All authors read and approved the final manuscript.


Acinetobacter baumannii, an emerging opportunistic pathogen associated with a wide spectrum of infectious diseases. The gene for A. baumannii EF-Tu was sequenced, and recombinant EF-Tu was purified for antibody development. EF-Tu on the bacterial surface and the outer membrane vesicles (OMVs) was revealed by immune electron microscopy, and its presence in the outer membrane (OM) and the OMV subproteomes was verified by Western blotting with the EF-Tu antibodies and confirmed by proteomic analyses. EF-Tu in the OM and the OMV subproteomes bound to fibronectin as detected by Western blot and confirmed by a label-free real-time optical sensor. The sensor that originates from photonic crystal structure in a total-Internal-reflection (PC-TIR) configuration was functionalized with fibronectin for characterizing EF-Tu binding. Altogether, with a novel combination of immunological, proteomical, and biophysical assays, these results suggest association of A. baumannii EF-Tu with the bacterial cell surface, OMVs, and fibronectin.A conundrum has long lingered over association of cytosol elongation factor Tu (EF-Tu) with bacterial surface. Here we investigated it with  Acinetobacter baumannii, has emerged as one of the most important nosocomial pathogens [ A. baumannii was identified in the US military personnel deployed to Iraq and Afghanistan [Acinetobacter infections are associated with natural disasters, such as the 1999 earthquake in Turkey [A Gram-negative and obligate aerobic bacterial species, athogens –4, raisihanistan . Intereshanistan . Besidesn Turkey  and the n Turkey .A. baumannii infections, little is known about the pathogenesis. To explore the fundamental mechanisms, we tested whether extracellular proteins of A. baumannii mediate the bacterial attachment [A. baumannii we isolated [A. baumannii was detected in cell-free cultures, the data suggesting release of EF-Tu from the bacterial cells. The release appeared unlikely to result from cell death and lysis but rather likely to be regulated, because the mutants, as viable as the wild type, exhibited deficiency in the release and cell adhesion [A. baumannii, is translation elongation as deduced from the E. coli EF-Tu, because EF-Tu and translation are highly conserved throughout the bacterial domain [·GTP serves as a carrier of codon-specified aminoacyl-tRNA to the ribosomal aminoacyl site. Eubacterial EF-Tus belong to the superfamily of GTP-binding proteins. It is not a membrane protein, since EF-Tu lacks a signal sequence and transmembrane domains that mediate protein translocation across cell membrane.For such austerity of tachment . Proteinisolated . With a adhesion . The EF-l domain –12. SpecEscherichia coli grown in the absence of sucrose [Neisseria gonorrhoeae [E. coli EF-Tu was detected again in the OM fractions of the cells adherent to abiotic surface [Staphylococcus aureus biofilm development [Lactobacillus johnsonii [P. aeruginosa [ Listeria monocytogenes in the infected host [This has led to a conundrum concerning EF-Tu release. The original clue to this question may come from a study with the sucrose-dependent spectinomycin-resistant mutants of  sucrose . EF-Tu worrhoeae . Two dec surface . The bacelopment , in mediohnsonii  or P. aeruginosa . The EF-ruginosa , as a tated host , and as ted host  and acquted host , 22.A. baumannii EF-Tu is associated with outer membrane vesicles (OMVs). The rationale is based on the proteomic analyses that have implicated EF-Tu association with OMVs in multiple bacterial species [A. baumannii [A. baumannii actually produces OMVs [How the surface-associated EF-Tu is released still seems to be an enigma. Our hypothesis was that  species  and withaumannii , and A. ces OMVs . To testA. baumannii  ATCC19606 strain was sequenced and the protein was purified for antibody development. The ATCC 19606 strain was chosen for novelty because its genome was not completely sequenced and the EF-Tu encoding gene was not studied at the time we started our investigation. The availability of genome sequencing data for the ATCC 17978 strain greatly facilitated our study. Based on the genome data, there are two genes for EF-Tu, namely tufAa and tufBa, both identical [tufAe and tufBe of E. coli. The tufAe deletion caused E. coli growth defect in rich media, while the tufBe deletion did not [tufAe is functional. These data led us to clone and sequence tufAa of the A. baumannii 19606 strain. Comparison of the tufAa sequences from 17978 and 19606 strains showed 99.8% identity; the small difference resulted from two nucleotide changes located in 1,032 and 1,137 —GCA of the 19606 strain but GCG of the 17978 strain—a silent mutation in the codon for alanine. The gene of the 19606 strain was cloned and His-tagged; rEF-Tu (48 kDa) was expressed and purified to homogeneity  and the OMV fractions (lane 2) in SDS-PAGE . EF-Tu win vitro trypsin proteolysis and capillary LC/MS/MS. The degraded peptide masses were determined and searched across the bacterial protein databases with the P < 0.05-based MOWSE scoring algorithm [While the evidence of Western blot for presence of EF-Tu in the OM and the OMV fractions appeared to be convincing, there were some drawbacks of Western blotting analyses, such as lack of scope due to limitation on utilizing costly arrays of antibodies to target multiple proteins. Proteomic analyses of the OMV and the OM subproteomes were carried out to address them. Briefly, total proteins in the lane of SDS-PAGE as shown in lgorithm . By thislgorithm . The conA. baumannii EF-Tu was detected in OMVs and on the bacterial cell surface  and the coating was detected by measuring the resonant wavelength shift; (iii) the subsequent washes removed the unbound protein molecules, leading to a minor reduction in the resonance shift, but the substantial changes still remained indicating the effective coating of the sensor by fibronectin. The binding thickness of fibronectin on the sensor was determined to be 4.8 nm corresponding to the 4.2 nm resonance shift calculated with a transfer matrix method [μg/mL, the resonant shift level changed from 0.8 nm to 1.1 nm , it seems possible that the proteins are hitched by DNA into OMVs or vice versa. Moreover, the presence of EF-Tu in both OMV and OM subproteomes is not just coincident but consistently documented [A. baumannii [N. meningitides [E. coli [A. baumannii OMV fraction [A. baumannii from clinical isolates, but we employed the standard ATCC19606 strain. They grew the culture under shaking condition while we used plates. While a combination of physical, immunological, and biochemical evidence appears to be convincing, we plan to compare data acquired from the standard and the clinical strains concerning the discrepancy.One of the intriguing observations is the presence of cytosolic proteins in the OMV and the OM subproteomes, for example, DNA binding proteins  in OMV and EF-Tu (#57) in the common subproteome. Considering OM's hydrophobic nature, we were tempted to suspect cytosolic protein contamination. Nevertheless, detection of DNA-binding proteins in OMVs seems unlikely to be attributed to contamination, as DNA-binding proteins and DNA were detected with in eae OMVs , 37. Sincumented . Its precumented . EF-Tu wcumented . EF-Tu waumannii . It alsongitides , 39 and [E. coli . Howeverfraction . This di L. johnsonii [P. aeruginosa [A. baumannii EF-Tu was detected in OMVs and on the bacterial cell surface, these data seem to support a notion that A. baumannii EF-Tu contributes to mediating adhesion of the bacterial cells and OMVs to macrophages through binding to fibronectin on the host cells, a hypothesis to be tested in the future. The role of the binding seems to be intriguing. EF-Tus ofohnsonii  and P. aruginosa  are invoruginosa . Given truginosa  and thattufAa encoding EF-Tu of A. baumannii was cloned by following the manufacturer instruction . Since the genome of the A. baumannii 19606 strain was not available when we conducted this study, tufAa of A. baumannii 17987 was used for designing specific primers and PCR was performed with the 19606 DNA template. Forward primer: 5′- GACGACGACAAGATGATGGCTAAAGCCAAG-3′ and the reverse primer: 5′-GAGGAGAAGCCCGGTCCGTCACTATATTATGCTTATGC-3′. The PCR product  was cloned into the pTriEX-4 Ek/LIC expression vector , which added an N-terminal hexa-histidine (6xHis) for purification by affinity chromatography and S-tag. The positive recombinant EF-Tu (rEF-Tu) clone DNA was verified by DNA sequencing analysis. The expressed rEF-Tu was fused with N-terminus His-S-tags containing 48 amino acids and purified by nickel affinity chromatography under native conditions by following the manufacturer protocol (Qiagen). The purified protein was analyzed with SDS-PAGE to determine the presence of rEF-Tu and was confirmed further by immunoblot by using monoclonal antibody to His-tag. The identity of rEF-Tu was further confirmed by N-terminal microsequencing as we described before [The gene μg rEF-Tu with complete Freund's adjuvant. Individual rabbits were boosted 3 times with the same amount of antigen in incomplete Freund's adjuvant at intervals of 21 days. Serum samples were collected and used in immunological studies. ProSci Incorporated provided us with sera prior to immunization and 3 bleed collected serums.The antiserum specific to rEF-Tu was developed by ProSci Incorporated. Briefly, rabbits were injected subcutaneously with 100–200 A. baumannii (ATCC19606) OMVs were isolated from LB agar plates as described previously [600 nm of 5. Then, bacteria were collected by low-speed centrifugation  for 5 min, and the recovered supernatant was centrifuged at 12,000 g for 10 min and further passed through the 0.2-μm pore size filters (Millipore). OMVs in the supernatant were then collected by ultra centrifugation at 100,000 g for 12 hours at 4°C and resuspended in PBS. OMPs were extracted according to Caldwell et al. [eviously  with modl et al. .A. baumannii cells and purified OMVs were immunogold-labeled with the anti-EF-Tu antibodies as described previously with some modifications [TEM was conducted according to a standard protocol , 45. Forications . BrieflyA. baumannii anti-rEF-Tu antibodies at a dilution of 1 : 3000 in 1% (w/v) blotto for 2 hrs at 25°C. The membrane strips then were washed, incubated in alkaline phosphatase-conjugated goat anti-rabbit antibodies , at a dilution of 1 : 5000 in 1% (w/v) blotto for 1 hr at 25°C, washed, and developed with 5-bromo-4-chloro-3-indolyl phosphate/Nitroblue Tetrazolium . For 2D gel electrophoresis, proteins from OMV and OM fractions were solubilized for isoelectric focusing (IEF) in 8 M urea, 2% CHAPS, 2% IPG buffer (Amersham Biosciences), 20 mM DTT, and traces of bromophenol blue. Sample was loaded on a 13 cm Immobiline DryStrip pH 3–10 (Amersham Biosciences) by rehydration and left under oil overnight. IEF was conducted under 17,000 Vh by using the Multiphor II (Amersham Biosciences) at 20°C. The IEF strips were equilibrated in Equilibration solution , and 0.02 M DTT) for 10 minutes. SDS-PAGE (10% w/v) was conducted as previously [ Proteins from OMV and OM fractions were separated on 10% SDS-PAGE  and staieviously . After seviously . Membran−1 DTT and incubating at 25°C for 5 min. The proteins (30 μg) were fractionated by SDS-PAGE . After staining, the proteins-containing lane in replicate was sliced into pieces (1 × 1 mm) for in vitro trypsin proteolysis. Capillary liquid chromatography-tandem mass spectrometry (LC/MS/MS) was performed, and the peptides derived from the proteins in the gel slices were determined with a linear ion trap tandem mass spectrometer in which the top 7 eluting ions were fragmented by collision-induced dissociation. Proteins were identified by following a standard protocol [P ≤ 0.05 in a search for random matches, and the proteins consistently detected in the replicates were counted. The analysis was performed as described previously , 48. Briprotocol , in whicprotocol  and the μg/mL for 24 hr at 4°C. Then, the membranes were washed and incubated with the rabbit antifibronectin antibodies at 1 : 10,000 dilution in 1% blotto for 2 hr at room temperature. Subsequently, the membranes were washed and incubated with the alkaline-phosphatase-conjugated goat anti-rabbit antibodies at a dilution of 1 : 20,000 in 1% blotto for 1 hr, washed, and developed as above. Nitrocellulose membranes transferred with proteins from OMV and OM fractions were blocked, washed, and incubated with pure fibronectin  at 10 μL, 200 μg/mL) on the sensor chip followed by incubation at 25°C for the indicated time as in μL PBS for measuring the thickness of bound protein. Finally, PBS was replaced with the analyte solution of EF-Tu or the proteoglycan 4 control in PBS . For the preparation of the control, the DNA (1.2 kb) encoding a portion of proteoglycan 4 was cloned; the polypeptide (44 kDa) was expressed and purified from baboon temporomandibular joint cells; it was tested negative in fibronectin binding by the same antibody-based assay as shown in The sensor originates from a prototype of a photonic crystal structure in a total-internal-reflection (PC-TIR) configuration . The desA. baumannii gene for EF-Tu , electrophoresis analysis of A. baumannii OMV DNA , as well as proteins present only in OMV (Table S1) and in OM subproteomes (Table S3), and proteins shared by OMV and OM subproteomes (Table S2).The Supplementary Materials include sequences of the Click here for additional data file.


The objective of this study was to describe a 12-year (1997–2008) observation of substance-related incidents occurring at rave parties in the Netherlands, including length of visits to first-aid stations, substances used, and severity of the incidents. During rave parties, specifically trained medical and paramedical personnel staffed first aid stations. Visitors were diagnosed and treated, and their data were recorded using standardized methods. During the 12-year period with 249 rave parties involving about 3,800,000 visitors, 27,897 people visited a first aid station, of whom 10,100 reported having a substance-related problem. The mean age of these people was 22.3+/−5.4 years; 52.4% of them were male. Most (66.7%) substance-related problems were associated with ecstasy or alcohol use or both. Among 10,100 substance-related cases, 515 required professional medical care, and 16 of these cases were life threatening. People with a substance-related problem stayed 20 min at the first aid station, which was significantly longer than the 5 min that those without a substance-related health problem stayed. These unique data from the Netherlands identify a variety of acute health problems related to the use of alcohol, amphetamines, cannabis, cocaine, ecstasy, and GHB. Although most problems were minor, people using GHB more often required professional medical care those using the other substances. We recommended adherence to harm and risk reduction policy, and the use of first aid stations with specially trained staff for both minor and serious incidents. Rave parties or house parties with DJ-directed, fast-paced electronic music and light shows were organized In the early 1990s, a new music culture called In the Netherlands, rave parties are allowed only if strict regulations are met. One of these is that a first aid station is required to take care of rave party attendees with various health-related problems. Here we present an overview of substance-related visits to first aid stations at rave parties in the Netherlands between 1997 and 2008.In 1997, 2001, 2005, and 2009, surveys of substance use in the general population in the Netherlands were conducted. Each of the substances covered in the present study were included in these surveys. During this period, lifetime prevalence of cannabis use generally increased (from 19.1% in 1997 to 25.7% in 2009), as did ecstasy use (from 2.3% to 6.2%) and cocaine use . For the first time in 2009, lifetime prevalence of γ-Hydroxybutyric acid (GHB) use was determined to be 1.3%, and the lifetime prevalence of alcohol use was 84% This was a prospective observational study of rave-party attendees who presented themselves for help at first aid stations at rave parties during the period 1997–2008. All persons seeking first aid were registered, but only those with substance-related problems were included in this study. Health-related incidents are described, together with length of medical care, severity of the incidents, predictability of symptoms, and short-term risks. Specifically, the following information was collected with regard to rave party attendees who sought help at first aid stations: (1) Length of stay, (2) substances used alone or in combination, and (3) nature of the substance-related problems.In an unpublished 1996 prospective pilot study of rave-party attendees, those seeking first aid were divided into two groups. The first was a self-care group. These people visited a first-aid station with only minor health-related problems and were not included in the study. The second group visited the first aid station seeking help or advice. For this group, a standard questionnaire was developed to ask about their health-related problems In the present observational study, data were collected prospectively and anonymously. According to Dutch regulations, neither medical nor ethical approval was needed to conduct the study. The study was not supported financially in any way. The data were obtained from files maintained by Educare, a nonprofit organization that provides first-aid assistance at large-scale events. The Educare Board of Directors consented to our using the data for scientific purposes.Upon entering the first aid station, the person was seen by a clerical officer, who determined whether medical assistance was necessary or self-care was sufficient. If aid was required, the person was referred to a member of the medical staff. This staff included qualified nurses, paramedics, and physicians, all of whom had received training in rave-related health risks, including the effects of psychoactive substances. They had also been trained to use the standardized questionnaire. An experienced co-worker was appointed to assist the staff in filling out the questionnaires, and this person coached all of the staff members in using the questionnaires. After the rave-party attendee had been discharged from the first aid station, the co-worker checked all of the data to verify their integrity.moderate or severe on the Emergency Severity Index The number of visitors to each rave party  was obtained from the organizers of the event. Serious health-related incidents were defined as those rated as r and Mann-Whitney U tests were used for the parametric and nonparametric data, respectively. A p value of <0.05 was the cut-off for significance. To explore relationships between health-related incidents and substance use, an logistic regression analysis was performed. A stepwise forward regression model was used, with P(in) = 0.05 and P(out) = 0.10; a maximum of 20 iterations was specified and a cut-off value of 0.5; the predictors of health-related incidents were added using a stepwise procedure. The models were evaluated for acceptable fit and proportions of variance explained. From each of the specified models, odds ratios >2 are presented. All analyses were performed using SPSS version 17.0.Descriptive statistics were used to understand the demographic characteristics of the sample and the nature of their substance-related visits to first-aid stations. To evaluate the statistical significance of the results, Person's N = 27,897; 0.7% of all visitors) of the people visiting a first-aid station presented with complaints that needed some form of medical attention. The mean age of all people visiting first-aid stations was 22.3 years (SD = 5.4), but the age of the visitors increased significantly from 18.7 years in 1997 to 24.0 years in 2008 . Visitors seeking help at first-aid stations were approximately equally divided between males (52.4%) and females (47.0%).From 1997 to 2008, 3,793,500 visitors attended 249 rave parties. Most (70%) of the raves occurred at night. Many (p<.001) , representing 0.3% of all rave party visitors, experienced a substance-related incident. The incidents were medical (80%), traumatic (9%), psychological (4%), or miscellaneous (7%). The median overall length of stay at a first aid station was 10 minutes, but it was 20 minutes for substance-using visitors and 5 minutes for nonsubstance-using visitors—a difference that is statistically significant (p<.001) .n = 6912, 64.4%) of the substance-using visitors reported using only one substance, which was usually ecstasy  or alcohol , but a substantial proportion  reported using two substances simultaneously. The most commonly reported combined use was ecstasy with alcohol   .The most common substance-related health problem was a general feeling of being unwell/fainting, which was associated with the use of all substances. Additional minor health-related problems were associated with the different substances. Using amphetamines was associated with having cramps (OR = 6.9); cocaine, with having a high body temperature (>37.5°C) (OR = 29.5) or palpitations (OR = 6.5); and GHB, with altered consciousness (OR = 32.7) . AlthougN = 55), with relative risk of 31.9 between 2001–2004 and 48.9 between 2005–2008  and GHB/ecstasy use  (N = 16) were substance-related; they included four cases of excited delirium, and three each of circulatory insufficiency, respiratory insufficiency, hyperthermia, and severe trauma.The number of rave-party visitors who sought first aid fluctuated during the 12 years of the study. Between 1997 and 2000, 7,136 people (1.0% of the visitors) needed first aid. Between 2001 and 2004, the number rose to 13,755 (0.8% of the visitors). From 2005 to 2008, 7,006 visitors sought first aid. Across the 12 years, a total of 515 cases were considered serious  . The comnd 44.8) –6. Finalnd 44.8) , N = 16 Approximately one-third of all rave-party visitors who sought first aid reported having a substance-related problem. Visitors with substance-related problems stayed longer at first aid stations than those without a substance-use problem. Altogether, 515 of 10,100 substance-related incidents were classified as serious, and 16 of these were life-threatening. Most substance-related incidents were associated with ecstasy or alcohol use or both. It is noteworthy, however, that in the Netherlands alcohol use is relatively common, but ecstasy use is not. It is possible that the willingness of rave-party visitors to present themselves at a first aid station with health-related complaints was related to the drug that they used. For example, ecstasy users' Unlike what most other recent studies from various countries have found In the current research, no evidence was found for life-threatening, acute effects of GHB. Nevertheless, professional medical care is often required after GHB use and the syndrome that can occur  can be dangerous. Health education should focus on these secondary effects in addition to the primary effects.Questions remain about whether the relatively low rate of severe incidents that occurred was related to the open nature and legal status of rave parties in the Netherlands. It would, therefore, be important to replicate this study in other countries. To our knowledge, there is no other published research on substance-related incidents that occur during large-scale events. It would be worthwhile for future research to focus on the causes of these incidents There were limitations of the current study that should be acknowledged. For example, long-term effects on substance use or drug addiction were not addressed. Although the study sample was large, it included only self-referrals, which might not be representative of all health-related incidents at rave parties. It is possible that many people who experienced negative effects did not present themselves at a first aid station. In fact, Wijngaart et al. and de Bruin et al. reported that some rave party visitors sought help from friends, security personnel, or food-service staff Only a small proportion of rave-party visitors (0.3%) reported substance-related health problems. The problems that were reported at first aid stations were usually related to ecstasy or alcohol use. Substance users who sought first aid stayed four times as long at a first aid station as nonsubstance users. A total of 515 of the substance-related incidents could be regarded as serious; this amounts to 0.01% of all party visitors, 1.8% of all visitors who sought first aid, and 5.1% of all substance users who sought first aid. Sixteen cases were classified as life threatening. Visitors who used GHB, with or without alcohol or ecstasy, and those who used cocaine were highest on relative risk of having a serious incident. Finally, it should be notes that although lifetime prevalence of GHB use is low, this substance causes many problems.


Clostridium difficile associated disease (CDAD) has risen in incidence and the experience in the US military has not been described.We evaluated the U.S. military’s database and identified CDAD cases and demographic characteristics among affected military personnel from 1998 to 2010.2,423 cases were identified. CDAD incidence was 13.2 cases (95% CI: 12.7-13.7) per 100 K p-yr and increased over study years. CA-CDAD and HA-CDAD incidence was 5.5  per 100 K p-y and 1.3  per 1,000 hospitalizations respectively. Females comprised a larger proportion of CA-CDAD than HA-CDAD  cases as did Air Force service . On multivariate analysis female gender, Coast Guard or Air Force service, and a married status was associated with CA-CDAD whereas Male gender and Marine Corps service were associated with HA-CDAD cases.CDAD has increased among military personnel, with female cases more likely to be community associated. Gender, marital status and branch of service had the strongest association with CDAD subtype. Further work is needed to evaluate the epidemiologic factors that have led to these increased rates in otherwise low-risk populations and associated sequelae. Clostridium difficile associated disease (CDAD) usually manifests as an inflammatory, cytotoxin-mediated enteric disease with a wide spectrum of severity which includes asymptomatic carriage, acute or persistent diarrhea, and fulminant colitis with sepsis . All identifiable information was removed and subsequently provided to study investigators.The study protocol was approved by the Naval Medical Research Center Institutional Review Board in compliance with all applicable Federal regulations governing the protection of human subjects. Datasets were de-identified and therefore a waiver of informed consent was granted by the Institutional Review Board.CDAD cases were identified by extracting individual encounters with ICD-9 codes for clinician diagnosed CDAD (008.45). Setting of infection, community vs. healthcare-associated, was defined using modified Centers of Disease Control and Society of Healthcare Epidemiology of America (CDC/SHEA) case definitions . CommuniDemographic comparisons between CDAD cases (CA- and HA-) were assessed using Chi-square test for categorical variables or Wilcoxon rank-sum (Mann-Whitney) test, as appropriate, for continuous variables lacking a normal distribution. Incidence rates and 95% confidence intervals were calculated using person-year denominator data for the study years for personnel in active duty service (CA) and hospitalizations (HA). Multivariate analysis of cross-sectional demographic and clinical variables associated with CA- vs. HA-CDAD was performed using log-binomial models to estimate prevalence ratios (PR). Covariates associated with CA- or HA-CDAD on univariate analyses were included in the final models using a stepwise selection approach with terms removed and added based on significance levels of 0.20 and 0.15 respectively.C.difficile diagnosis.Re-hospitalization rates were calculated by identifying inpatient encounters for C. difficile disease which occurred after 7 days and within 60 days from the original All statistical analyses were performed using SAS v. 8.2 for Windows  and Stata version 12  for Windows and statistical significance was evaluated using an alpha = 0.05.A total of 2,423 active duty service members were diagnosed with CDAD  over the study period for an overall incidence of 13.2 cases/100 K p-yr . The baseline characteristics of cases are shown in Table Females constituted a larger proportion of the CA-CDAD cases than HA-CDAD cases . In terms of branch of service, the majority of total and HA-CDAD cases came from the Army, while the Navy and Coast Guard cases were evenly distributed among HA- and CA-CDAD cases, and CDAD was more common in Air Force members . Among Marines, HA-CDAD occurred more commonly . There were no significant differences in the age, military rank or race of the CDAD cases. The duration of follow-up (end of active duty service) was longer for those with CA-CDAD than those with HA-CDAD.The overall incidence Table  of CA-CDDuring the study period, the overall incidence of HA-CDAD was1.3  cases per 1,000 hospitalizations  were inpatients at the baseline visit and for 728 of those  the baseline visit represented their only hospitalization on record. Among all HA-CDAD cases, if a prior admission was present it most often occurred in the 1 month prior to the CDAD diagnosis. Specifically, for outpatients and inpatients at the baseline visit, the median number of days from the last admission was 20 days  and 18 days  respectively (data not shown).Table Among all CDAD cases, 1,451 (60%) subjects had subsequent CDAD-related medical encounters after the initial visit and of these, 582 (40%) had two or more visits during the follow up period. For a majority of both CA- and HA-CDAD cases, the last CDAD-related code occurred within a year after diagnosis. Of the 1,018 subjects identified as CA-CDAD cases, 519 had subsequent CDAD coded visits with the majority occurring within a month of the original visit, and 43 (4.2%) and 64 (6.3%) having a CDAD visit coded after 8 weeks and 4 weeks respectively. Among the 1,405 HA-CDAD cases, 932 had subsequent CDAD coded visits the majority of which also occurred within a month of the original diagnosis. For HA-CDAD cases, 68 (4.9%) and 140 (9.9%) had CDAD visits after 8 and 4 weeks, respectively, from the original diagnosis , 100 (4.3%) subjects had an inpatient encounter within 2 months for CDAD. Ninety-six of the admissions were among cases previously classified as HA-CDAD, whereas only 4 cases were among the previously classified as CA-CDAD  all appear to contribute to recurrent disease ,18. In oOur study has several strengths and limitations. Access to the large, worldwide deployed, US military population allowed us to select and follow a large number of CDAD cases from all services and a variety of clinical settings but for which access to care, testing and therapy is relatively uniform. This study represents the most comprehensive and extensive review of CDAD in the military population and reveals the epidemiology of CDAD largely has paralleled the trends seen in other civilian and older populations. A main limitation of our study is the medical encounter database nature of its design and reliance on ICD-9 coding which may increase the risk for misclassification. However, recently published validation studies in inpatient populations have noted a single CDAD ICD-9CM code has a high specificity for CDAD disease  and may therefore be adequate tools for surveillance . One conMultiple factors, including period effects, may have influenced the changes in CDAD rates seen in our study. Military specific factors, such as demographic transitions in the military population, and the effect of operational requirements with attendant changes in overall exposure to combat deployments may have influenced rates in our population during this time period but it is likely that most of these effects would have been limited. Interestingly, service in the Army and Marine Corps, younger, an enlisted populations are more heavily involved in combat operations, and were also more likely to be associated with HA-CDAD. In addition, other risk factors and secular trends, which have been documented from other populations , may also have influenced the rates and increases seen in our population.In summary, in this retrospective study of the generally younger, active duty U.S. military population, our data documents a notable rise in incidence of both CA-CDAD and HA-CDAD, similar to what has been seen in other populations. Of note, we documented a female association with CA-CDAD which persisted when controlling for service branch and low rates of recurrence or re-infection after the index visit. Incidence and risk factors for CDAD among military personnel likely differ from those of the general population, and our findings regarding the incidence and patterns of CDAD need further study.None of the authors have a conflict of interest or a financial disclosure related to this work which was performed as part of official duties.RLG, CKP and MKR participated in the data analysis and drafting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/609/prepub


Consistent with the lesion data, single-unit recordings from the PL showed that the majority of CS-responsive neurons fired at a higher rate in the fearful context than in the safe context, paralleling the behavioral discrimination. Taken together, the current results suggest that the PL is involved in selective expression of conditioned fear to an explicit (tone) cue that is fully dependent on contextual information.The ability to regulate emotional responses in various circumstances would provide adaptive advantages for an individual. Using a context-dependent fear discrimination (CDFD) task in which the tone conditioned stimulus (CS) is paired with the footshock unconditioned stimulus (US) in one context but presented alone in another context, we investigated the role of the prelimbic (PL) cortex in contextual modulation of the conditioned fear response. After 3 days of CDFD training, rats froze more to the CS presented in the fearful than in the safe context. Following bilateral lesions of the PL, rats showed similar levels of freezing to the CS in both contexts, in contrast to the sham-lesioned control animals. The lesions did not impair the rats' ability to discriminate contexts  Learning to express context-appropriate fear responses constitutes an important survival strategy since a cue can signal danger in one context but not necessarily in another. Most forms of defense reaction impose heavy costs on the animal, consuming resources and sacrificing opportunities to perform other behaviors  area, a subregion of the mPFC, receives a number of afferent projections from the hippocampus and multiple sensory cortical areas and sends efferent projections to the amygdala  task, modified from a discrimination task previously used to demonstrate a contextual control of fear response in rats  guidelines.Male Sprague Dawley rats  were housed individually on a 12-h reversed light/dark cycle (lights on at 9:00 P.M.) with Rats were fully anesthetized using sodium pentobarbital  and mounted on a stereotaxic apparatus . The scalp was incised, and small holes were drilled for electrode insertion. For electrolytic lesions, stainless steel electrodes (0.3 mm in diameter) insulated with epoxy, except for 0.5 mm at the tip, were lowered bilaterally into the PL . The lesion was made by passing anodal current (1.0 mA for 15 s) at each site. Sham animals were treated in the same manner except that no current was passed.Under anesthesia, one or two bundles of eight fine wires  aimed at the PL , were lowered uni- or bilaterally. The tips of the electrodes were cut and gold-plated to yield impedance of 200–500 kΩ at 1 kHz. The electrode assembly was secured to the skull with six anchoring screws and dental cement. Four of the skull screws were used as the ground. Following surgery, rats were allowed to recover for at least 6 days before the experiment started.For the CDFD test, two contexts (Context A and B) with distinctively different configurations, odor, and color were used for training and testing. In addition, a novel context (Context C) was used for additional testing. Context A (fearful) and B (safe) consisted of one of the following context combinations : (i) a black Plexiglas box (30 × 25 × 25 cm) and citrus odor under blue illumination or (ii) a transparent Plexiglas box (27 × 25 × 34 cm) and mint odor under red illumination with an inverted V-shaped foam board inserted below the ceiling. Context C consisted of a white Plexiglas cylinder (25 cm in diameter) with cinnamon odor under white illumination , placed in a room surrounded by a black curtain and illuminated with indirect halogen lighting, was used for the open field test. The center area was defined as a square section in the center (46.2 × 46.2 cm). A video camera and a webcam monitored and recorded the rats' spontaneous activity. The movement trajectories were recorded and analyzed with an automated tracking system .For the PPI test, a rat was confined in a Plexiglas cylinder  and placed on a platform supported by a load cell . A small speaker  delivered the prepulse stimulus . The prepulse was followed by a startle stimulus  delivered through a tweeter . Vertical displacement of the load cell caused by the animal's startle reaction was amplified (×1000) and digitized by a DIO card . Stimulus presentation and data acquisition were automatically controlled by a custom program written in LabVIEW (NI). The test was performed inside a sound attenuating chamber (50 × 50 × 50 cm) to attenuate outside noise.n = 8) and the other half received sham lesions . After 7 days of recovery, testing was conducted in Context A and then in Context B, separated by 1 h. Three CS-only trials were presented to the rats in each testing session. The order of exposure to the context was counterbalanced across animals: some rats were tested in Contest A then in B, while others were tested in B then in A. On the following day, all animals were tested in novel Context C, to which they had not been previously exposed. Three minutes after they were placed in Context C, they were given 3 CS-only trials. Freezing was analyzed during the first CS presentation period in each context. For the recording experiment, they were given 6 CS-only trials in each testing session.As shown in Figure n = 8) or sham lesion (n = 8), followed by 7 days of recovery. They were tested in Context A and then B, or vice versa, separated by 1 h. Each test session lasted for 13 min per context. Freezing during the first 3 min was included for the analysis.All procedures were identical to those used in CDFD, except that no discrete CS was provided throughout the task. After habituation and training sessions, rats also received a PL , and a no-stimulus trial. A total of 60 trials were randomly presented, separated by random intervals (5–25 s). The PPI was calculated as follows: PPI (%) = 100 × (SS-PS)/SS, in which SS denotes the average startle amplitude to the startle stimulus, and PS denotes the average startle amplitude to the prepulse–startle stimulus.The single-unit activity was amplified by a head-stage amplifier (unity gain) and a main amplifier , filtered between 600 and 6000 Hz, and sampled at 32 kHz using a Cheetah data acquisition system . The digitized activity was stored in a personal computer for offline analyses.Z-scores were calculated for each unit and averaged. A cell-by-cell analysis was performed by computing t-values based on paired comparisons between the matching bins. There were thirty 1-s bins for the duration of the tone CS, and the t-values were calculated by summing the Z-score differences (Context A vs. B) for each bin.Freezing, defined as the absence of movement except for respiration  compared the freezing levels and unit responses to the CS in Context A to those in Context B (and Context C). Following the termination of the behavioral testing and/or recording, the rats were overdosed with pentobarbital sodium . The electrode placements were marked by passing anodal current (10 μA for 10 s) through the tip of 2 or 3 electrodes in each bundle. Animals were then perfused with saline (0.9%) and paraformaldehyde (10%). The brain was removed, post-fixed overnight, and transferred to a 30% sucrose solution for 2–3 days. The tissue was sectioned transversely at a thickness of 50 μm. All sections were stained using cresyl violet following a reaction in a 2% potassium ferrocyanide solution to visualize iron deposits created by the lesion. The stained brain sections were analyzed using NIH Image (V.1.38) in order to quantify the extent of the lesions. The size of the lesion was calculated by dividing the lesioned area by the total area of the region of interest  identified from the atlas devised by Paxinos and Watson . For theAs shown in Figure F = 0.005, p > 0.9; day × group interaction, F2, 28 = 0.251, p > 0.7; context × group interaction, F1, 14 = 0.057, p > 0.8; day × context × group interaction, F2, 28 = 0.190, p > 0.8], the two groups (SHAM and PL lesion) were collapsed for the training data. All rats showed differential response following 3 days of training  and 3 (p < 0.0001). Seven days after surgery, the rats were tested in Context A, and then in B  on the same day. On the following day, the rats were tested in Context C to determine whether the fear CR re-appears to the CS in a novel context. Figure F2, 28 = 8.661, p < 0.001], showing that the rats froze more in Context A and C than in Context B. In addition, there was a significant group effect  as well as a context × group interaction , indicating that the SHAM animals froze more to the CS than PL lesion animals in Context A (p < 0.01), as confirmed by the post-hoc test. Further post-hoc tests revealed that the SHAM rats froze more to the CS in Contexts A and C than in B  and more in Context A than in C (p < 0.05). In contrast, PL lesion rats maintained a similar level of freezing throughout the three test sessions . These results indicate that the post-training PL lesion disrupted the CDFD and renewal of the fear response to the CS in a novel context.For the CDFD, the rats received 3 CS–US pairings in the fear context and 10 CS-only trials in the safe context. This discriminatory training was repeated for 3 days. Because a RM ANOVA revealed that there was no difference between groups in their freezing during training . The difference did not reach significance because some of the SHAM rats exhibited ceiling levels of pre-tone context freezing. However, in Context C where the rats' freezing was almost absent, the difference between freezing to the tone CS and that to the context was significant , indicating that the CS-evoked freezing is a dominant response in CDFD.Since the focus of the current study is the tone-evoked response, it needs to be determined whether the level of freezing discriminated the discrete tone CS from the background context. Therefore, based on the data from the SHAM group, we tested whether freezing to the tone CS in Context A  = 0.412, p > 0.6, Figure t(14) = 0.362, p > 0.7, Figure t(14) = 0.204, p > 0.8, Figure To test the effect of the PL lesion on ambulatory and emotional response, the open field test was conducted following completion of the CDFD task. There were no significant differences between the SHAM and PL lesion groups in total distance  and day × context interaction . A post-hoc analysis revealed that the rats froze more to the CS in Context A than in B on training days 3 (p < 0.01) and 4 (p < 0.05)  Figure . Twenty-) Figure  as evidet-test, p > 0.2; 6.09 Hz ± 1.42, for Context A; 7.56 Hz ± 1.81 for Context B). The spontaneous firing rate of PL units collected in the present study was comparable to that from previous studies  = 2.809, P < 0.01]  were short-latency CS-responsive. Because context modulation of the CS was the primary interest of the current study, we mainly analyzed the CS-responsive units. To reveal any context-induced changes in spontaneous activity, firing rates during the 3-min baseline period were compared. The average firing rates varied (0.31–34.60 Hz) but did not differ significantly between the 2 contexts  = 2.37, p < 0.05, Figure t-test, t(23) = 2.96, p < 0.01, Figure t-values based on paired comparisons between the matching bins. There were thirty 1-s bins for the duration of the tone CS and the t-values were calculated by summing the Z-value differences (Context A vs. B) for each bin. Among the units that have modulated their firing rates significantly during the presentation of the CS, 46% (11/24) showed significant positive t-values, whereas only 13 % (3/24) showed significant negative t-values. A chi-square test revealed that a higher percentage of tone-responsive neurons have positive t-values than negative t-values . We found 33% of PL units 24/72) were PFUs. Figure 2 were PFZ-scores in each phase. We found no difference between the two phases. Specifically, the Z-scores significantly differed between the two contexts for both the short-latency response  and sustained response  data , but there was no phase effect  or context × phase interaction . Note that 5 out of 27 units analyzed for the 50-ms bin data did not fire during the pre-CS baseline (1 s) throughout the initial or last 3 trials and were excluded from this analysis. These data exclude the possibility that a short-term habituation-like process across 6 trials during the recording session might have reduced the PL activities implicated in context-specific fear expression.Generally, fear responses decrease when the subjects are presented with repeated CS-alone trials, indicating within-session habituation which eventually contributes to extinction  as shown in Figure The low level of fear response in Context B developed gradually, resembling the course of extinction learning in which an initially high level of fear response declines over repeated presentation of the CS without the US. We observed that the level of freezing to the CS in both contexts was equally high at the beginning of training Figure . These dOne might pose a possibility that the lesions extending to Cg or IL might have contributed to the deficits in CDFD performance. However, it is unlikely that the partial lesion in those areas would have attenuated the discriminatory response because the lesion effect observed in our study contracts what might be expected from Cg or IL lesion. For example, IL lesion or inactivation facilitated recovery of extinguished fear response  but also in Context C  without a significant change in freezing in Context B (inhibitory context) supports this interpretation. Normally, the inhibition of the conditioned response formed as a result of extinction training is vulnerable to context shifts Bouton, . The curInteresting parallels should be noted between the current results and a study employing appetitive discrimination tasks. Haddon and Killcross  trained N-methyl-d-aspartic acid receptor-dependent manner , which recruits higher executive function of cortical areas. Therefore, lowering the activity of a human brain area that corresponds to the PL might effectively alleviate the symptoms of fear-related disorders.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Legionella pneumophila (LPN) can cause a lethal infectious disease with a marked inflammatory response in humans. However, the mechanism of this severe inflammation remains poorly understood. Since necrosis is known to induce inflammation, we investigated whether LPN induces necrosis in macrophages. We also analyzed the involvement of lysosomal cathepsin B in LPN-induced cell death.The human monocytic cell line THP-1 was infected with LPN, NUL1 strain. MG132-treated cells were used as apoptotic control cells. After infection, the type of cell death was analyzed by using microscopy, LDH release and flow cytometry. As a proinflammatory mediator, high-mobility group box 1 (HMGB-1), was measured. Cathepsin B activity was also measured and the inhibitory effects of cathepsin B on LPN-induced cell death were analyzed.THP-1 cells after treatment with high dose of LPN showed necrotic features with releasing HMGB-1. This necrosis and the HMGB-1 release were inhibited by a specific lysosomal cathepsin B inhibitor and were characterized by a rapid and high activation of cathepsin B that was not observed in apoptotic control cells. The necrosis was also accompanied by cathepsin B-dependent poly(ADP-ribose) polymerase (PARP) cleavage.L. pneumophila rapidly induces cathepsin B-dependent necrosis in a dose-dependent manner and releases a proinflammatory mediator, HMGB-1, from macrophages. This report describes a novel aspect of the pathogenesis of Legionnaires' disease and provides a possible therapeutic target for the regulation of inflammation.We demonstrate here that  Legionella pneumophila is an intracellular pathogen that causes rapidly advancing pneumonia and is sometimes life-threatening. After inhalation into the lung, the organism initially infects alveolar macrophages and replicates in these cells. The infected macrophages produce cytokines such as IL-β and TNF-α that activate both themselves and other immune cells [ne cells . HoweverShigella [Salmonella [Mycobacterium tuberculosis [L. pneumophila has been shown to induce apoptosis in macrophages or monocytic cell lines when the cells were infected at a low dose of bacteria [L. pneumophila, in which a high dose of bacteria was used [In infectious diseases, cell death that occurs as a result of interactions between the infectious organism and the host cell can have important implications for host defense or bacterial survival. Apoptosis is a typical programmed cell death that is tightly regulated by various proteases, requires ATP and does not involve inflammation . In contShigella , Salmonelmonella  and Mycorculosis  and the bacteria -9, inducwas used ,11.Recent research has implicated lysosomal function in cell death . Many tyL. pneumophila induces necrotic cell death in a monocytic cell line and in murine macrophages by comparing cell death induced by L. pneumophila with that induced by an apoptotic agent. We also examined the role of lysosomal enzymes in L. pneumophila-induced cell death. We found that potent activation of cathepsin B leads to necrosis accompanied by inflammation in cells infected with a high dose of L. pneumophila. In addition, cell death and inflammation were inhibited by attenuation of cathepsin B.In this report, we determined if PARP antibody was from Cell Signaling Technology  and anti-cathepsin B antibody (CA10) was from Abcam . CA074Me and zVADfmk were obtained from the Peptide institute .L. pneumophila NUL1 bacterial strain, serogroup 1, which was clinically isolated from the sputum of a patient at Nagasaki University Hospital [The Hospital , was useFemale A/J mice, 6-8-weeks old, were purchased from Japan SLC, Inc. . All animals were housed in a pathogen-free environment and received sterile food and water in the Laboratory Animal Center for Biomedical Science at Nagasaki University. Animal care and experimental procedures were performed in accordance with the Guidelines for Animal Experimentation of Nagasaki University with approval of the Institutional Animal Care and Use Committee.5 cells/well for all experiments except for fluorescent microscopy. After addition of L. pneumophila or MG132, the plate was centrifuged at 1000 × g for 5 min. For inhibition studies, the cells were pre-treated with CA074Me for 30 min before stimulation.The human monocytic cell line THP-1 was cultured in RPMI1640 medium containing 10% FBS. Murine alveolar or peritoneal macrophages were obtained from bronchoalveolar lavage fluid or peritoneal lavage fluid 3 days after injection of 4% thioglycollate into the peritoneum, respectively. The cells were plated into 96-well plates at a density of 1 × 10Cell death was evaluated using a cytotoxicity detection kit  based on the measurement of LDH according to the manufacturer's instructions. To determine cytotoxicity, the value of samples from 1% Triton-X-treated cells was defined as 100% cell death.g for 1 min, fixed onto slides and were then stained with Diff-Quik . Cellular morphological features were categorized by two cytologists. One hundred cells were counted three times and the results are presented as an average value.Cells were stained with 0.2% trypan blue and trypan blue-positive and -negative cells were counted. To observe morphological findings, the cells were centrifuged at 1,000 × 3 cells/well  or 1 × 105 cells/well  and were cultured in fresh medium overnight. After treatment, the cells were incubated with PBS containing 7.5 μg/mL Hoechst 33342 and 2 μg/mL PI for 30 min. The preparation was observed using a Leica DM400B fluorescent microscope .Mice macrophages were plated into a CultureWell chambered coverglass  at a density of 1 × 10Cells were washed and homogenized at 4°C in lysis buffer as described previously . Cell ly-, PI-), early apoptotic cells  and late apoptotic or necrotic cells . Cells were harvested after treatment, and 104 cells per sample were analyzed using a FACSCalibur flow cytometer and CellQuest software .To evaluate cell death changes, cells were simultaneously stained with FITC-conjugated Annexin-V and the non-vital dye PI  to enable discrimination of intact cells  were resolved by electrophoresis on a 12.5% polyacrylamide gel, and were transferred to a polyvinylidine difluoride membrane. After blocking the membrane in 10% FBS and 0.1% Tween-20 in Tris-buffered saline for 1 h at room temperature, blots were hybridized with primary antibodies overnight at 4°C. After hybridization with a secondary antibody, the immunocomplexes were visualized using an ECL Western Blotting Detection System .The concentration of extracellular HMGB-1 was measured using an HMGB-1 ELISA Kit II  according to the manufacturer's instructions. Briefly, 10 μL of samples were added to an anti-HMBG-1 antibody-coated plate. After incubation at 37°C for 24 h, the plate was incubated with peroxidase-conjugated anti-HMGB-1 monoclonal antibody for 2 h at room temperature. Color was developed using a 3,3',5,5'-tetramethylbenzidine and peroxidase solution. Absorbance was read at 450 nm.Cytotoxicity, morphological classification and HMGB-1 concentrations were expressed as means ± SD. One-way analysis of variance was used to determine statistically significant differences between groups. Tukey's test was used to confirm differences between individual groups.L. pneumophila used were calculated based on the ratio of bacteria to cells, and are expressed as multiplicity of infection (MOI). First, we assayed the time course of lactate dehydrogenase (LDH) release into the culture medium following incubation of THP-1 cells with various levels of L. pneumophila , in a dose- and time-dependent manner but not an apoptosis inducer, MG132 [L. pneumophila, the cells showed characteristic features that are consistent with necrosis such as the loss of plasma membrane integrity and irregular and poorly-stained nuclei in contrast to MG132-treated apoptotic cells : high-mobility group box 1; (PARP): poly(ADP-ribose) polymerase; (LDH): lactate dehydrogenase.The authors declare that they have no competing interests.YM designed the study, carried out the molecular studies, the cell culture, the animal study, image analysis and data analysis, participated in the flow cytometry and drafted the manuscript. HH carried out the flow cytometry. KY, HH, YYamad and SKa participated in the design of the study and revised the manuscript. SN, MS, KI, HK, YYamam and SKo coordinated the study. All authors read and approved the final manuscript.


S-transferase π (GST-π) are not only drug-resistance markers, but also prognostic markers of various cancers. The aim of the present study was to investigate the clinical significance of P-gp and GST-π in gallbladder carcinoma (GBC). Tissue samples from 42 patients with GBC were immunostained. Demographic, clinical and follow-up data were collected and analyzed. The positive expression rates of P-gp and GST-π in the GBC tissues were significantly higher  than that of chronic cholecystitis specimens  , and correlated with the Nevin stage of GBC. Multivariate analysis demonstrated that patients with positive expression of P-gp and GST-π showed a significantly lower 2-year survival rate  compared with patients with negative expression  . P-gp was also found to be an independent prognostic marker of 2-year survival rate by logistic regression analysis . Results of this study suggest that P-gp is a prognostic marker of GBC and the detection of P-gp and GST-π may contribute to the prognosis of GBC and the application of chemotherapy as a therapeutic treatment.P-glycoprotein (P-gp) and glutathione  Gallbladder cancer (GBC) is a common and lethal cancer with a poor prognosis due to its insensitivity to radioactive and chemical therapies. The 5-year survival rate of GBC is <5% ,2. EarlyP-glycoprotein (P-gp) is encoded by the multidrug resistance 1 (MDR1) gene and is a membrane glycoprotein. It acts as an energy-dependent drug efflux pump and is important in pharmacokinetics. Overexpression of P-gp is a major mechanism of drug resistance , thus, PS-transferase π (GST-π) is a subclass of GSTs, a polymorphic supergene family of detoxification enzymes involved in the metabolism of numerous potential carcinogens  between January 2000 and July 2005. Following excision, samples were paraffin-embedded. The diagnosis of GBC was established by histopathological analysis and surgery was performed according to the stage defined by the Nevin classification . In addiDemographic and clinical data, including age, gender, the presence of gallstones and disease history, were obtained. The patient cohort included 26 females and 16 males and the mean age at surgical resection was 60.81±1.30 years. All patients did not present with complications, such as hypertension, diabetes and chronicle hepatitis. Histological types of GBC were classified according to previous studies ,16, whicThis study was approved by the Ethical Committee of Xi’an JiaoTong University. All patients received oral and written information regarding the study protocol and signed an informed consent prior to inclusion in the study.Paraffin-embedded tumor tissues were sliced into 5-μm thick sections and mounted on glass. Slides were deparaffinized and rehydrated in 10 min through a graded alcohol series to deionized water in 1% Antigen Unmasking Solution  and microwaved to enhance antigen retrieval. Tissue samples were sequentially incubated with anti-mouse immunoglobulin coupled to horseradish peroxidase (HRP). Slides were incubated with the specific primary anti-GST-π  and anti-P-gp  monoclonal antibodies with an HRP-conjugated secondary antibody , and then stained with 3,3-diaminobenzidine and counterstained with hematoxylin and eosin. In addition, 10 tissue samples from patients with chronic cholecystitis obtained by cholecystectomy were used as the control group. Two pathologists independently observed and interpreted the results of the immunohistochemical staining.Staining of P-gp and GST-π was evaluated according to the percentage of positive cells under an optical microscope . Staining intensity was classified as the following: Negative (−), no immunopositive staining or <10% of positive cells observed; weak to moderate (+), 10–30% positive cells; and high (++), >30% positive cells.Patients were advised to undergo 2-year regular follow-ups following GBC diagnosis. Follow-up data from 36 patients were obtained and the remaining data were lost.2 test. The Fisher’s exact test was used to assess the differences between the positive rates when the number of total cases was <40. All statistical tests were two-sided. To elucidate the risk factors for prognosis , multivariate analysis was performed using the logistic regression model. P<0.05 was considered to indicate a statistically significant difference.Statistical analysis was performed using SPSS software . Differences of expression rate among groups were analyzed by Pearson’s χP-gp and GST-π were mainly expressed in the cytoplasm or membrane of GBC cells . The pos2=3.09; P=0.044)  were lower  than that in the later stages (IV and V)   . As NeviP=0.044) .2=−2.76, P=0.061)  was higher than that in the P-gp-positive patients (11.1%) (P=0.013). Similarly, 2-year survival rate in the GST-π-negative patients (45.5%) was also higher than that in the GST-π-positive patients (12.0%) (P=0.036). In addition, coexpression of P-gp and GST-π demonstrated the lowest 2-year survival rate (4.3%) (P=0.001) . P-gp waP=0.061) .2=0.20; P=0.003) and the coexpression rate was 59.5% (A significant positive correlation was also found between the expression levels of P-gp and GST-π in tumor tissues (Ras 59.5% .In the present study, expression levels of P-gp and GST-π in malignant lesions were found to be higher than that of benign lesions, indicating multiple drug resistance of GBC. In addition, majority of positive cells were located on the mucosal surface of the gallbladder, this phenomena was consistent with the role of P-gp as a multidrug transporter and support the mechanism of GBC. Similar results have also previously been identified suggesting that P-gp is substantially expressed on the biliary surface of hepatocytes and small biliary ductules .in vivo by reactive oxygen species and its induction represents part of an adaptive response mechanism to chemical stress caused by electrophiles (Although P-gp and GST-π correlate with drug resistance, their mechanisms and drug-resistant spectrum are different. GST-π is regulated rophiles . The drurophiles ,20. TherIt has been previously suggested that aromatic compounds can induce GST-π expression ,22. In aThe main prognostic factors of GBC are the clinical or pathological stages  and the A close correlation between GST-π and P-gp expression was also identified in the current study, with coexpression observed in 59.5% of patients with GBC. Similar studies have shown that the rate of coexpression was 93% in patients with leukemia and 80% in patients with lung cancer ,30. It hIn conclusion, results of the present study suggest that P-gp is a prognostic marker for GBC. In the future, the detection of P-gp and GST-π in patients with GBC may contribute to chemotherapeutic and surgical decisions.